Preparation of pure dimethyl benzimidazol cobamide adenosine coenzyme



May 5, 1964 G- NOMINE ETAL l PREPARATION OF PURE DIMETHYL; BENZIMIDAZOL 32'135 COBAMIDE ADENOSINE COENZYME Filed Jan. 21, 1963 SOLUBILITY OF THE PHENOLIC COMPLEX 0F 00 mum" CONCENTRATION OF PHENOL IN GRAMS PER IOOCIB muax gg m LUCIEN ASSE Y PIERRE BARTHELEMY #wdzw ATTORNEYS United States Patent 3,132,135 PREPARA ion on roan nnrnrnvi unmanne- AZOL CGEAMIDE ADENQSENE ENZYME Gerard Nomine, Noisy le Sec, Lucien Penasse, Paris, and

Pierre Bartheiemy, Clichy-sous-Bois, France, assiguors to Roussel-Uclaf, 8A., Paris, France, a corporation of France Filed Jan. 21, 1963, Ser. N 252,667 Claims priority, application France Jan. 22, 1962 6 Claims. (Cl. 260211.5)

vitamin B family has been determined by Barker et a1.

(J. Biol. Chem, vol. 235, 1960, p. 18-1). The recovery of dimethyl benzirnidazol cohamide adenosine coenzyme from a culture broth of Clostridium tetcmomorphum con-' sists of extracting the coenzyme from a previously concentnated aqueous solution with aqueous phenol, extracting the aqueous phenol extract with a mixture of acetone, water and ether followed by fractionation through a Dowex 5=0 resin column and a second phenol extraction.

recovery process is not only laborious but also results in a considerable loss in the coenzyrne.

It is an object of the invention to provide a novel economicalptocess tor the recovery of pure dimethyl benziniidazol 'cobamide adenosine coenzyme.

'It is another object of the invention to provide novel hydrated crystalline complexes of dimethyl benzhnidazol cob-amide adenosine lcoenzyme and phenolic compounds.

These and other advantages and objects of the invention will become obvious from the following detailed description.

The process of the invention for the recovery of pure dimethyl benzimidazol oobamide adenosine coenzyme comprises adding to an aqueous solution having a mini 'mumooncentnation of 0.05% of dimethyl benzirnidazol cobarnide adenos-ine coenzyme an amount of a phenol having the formula wherein R is at least one member selected from the group consisting of hydroxy, lower alkyl and hydnoxy lower allcyl and x is an integer from 0 to 2 less than its solubility limit in water to form a hydrated crystalline complex of dimethyl benzimidazol cobamide adenosine ooenzyme and the phenol having the formula wherein R is the phenolic compound, 11 is an integer from 1 to 3 and m is an integer from 2 to 10, redissolving the hydrated crystalline complex in an aqueous solvent and 3,132,135 Patented May 5, 1964 2 to. 3% of phenol, about 5% of resorcinol or hydroquinone and about 4% ofsaligenol.

The aqueous solutions of dimethyl benzimidazol cobamide adenosine coenzyrne to which the phenolic oompound tobe added must have a concentration of at least 0.05% of the said coenzyrne. If'the coenzyme concentration is below this value, the hydrated crystalline complexes not form due to its solubility in aqueous solutions in this range. The said aqueous solutions can be obtained by preliminary pmocessing by extnaction and purification of culture growths of microorganisms.

The hydrated crystalline complexes of dimethyl benzimidazol 'cobamide adenosinecoenzyme-phenolic compound oorresp 0nd to the generic formula 'zz loo n ia )n' 2 ')m wherein n is an integer from 1 to 3, m is an integer from 2 to 10 and R is a phenol having the tomnula wherein R is at least one member of the group consisting of hydroxy,=lower alkyl and hydroxy lower alkyl and x is an integer item 0 to '2; R is preferably hydroxy, methyl and/ or hydroxymethyl.

The hydrated crystalline complexes are slightly soluble in cold water but are quite soluble in aqueous solvents, such as aqueous acetone, aqueous dioxane or aqueous tetnahydrofunan at 50 C. Dimethyl benzimidazol cobamide adenosine coenzyme can {be precipitated in a pure state from these solutions by the addition of a water miscible solvent, such as acetone or dioxane or rtetrahydrofunan and ethanol in yields of better than 80% Dimethyl benzimidazol oobamide adenosine coenzyme is active in catalyzing the conversion methyl aspamtate and mesaconate.

Referring now to the drawing- FIG. 1 is a graph showing the solubility of the complex of phenol and dimethyl benzimidazol coharnide adenosine ooenzyme in aqueous solutions of phenol.

In the following examples there are described several pneferred embodiments to illustrate the invention. However, it should be understood that the invention is not intended to be limited to the specific embodiments.

EXAMPLE I Recovery With Phenol Step A.-Formati0n of .the hydrated phenol complex: 112 mg. of dimethyl benzimidazol cobamide adenosine coenzyme, being 89.5% coenzyrne and 9.9% Water were dissolved in darkness in 6 cc. of distilled water. 3 cc. of an aqueous solution of 6% phenol were added to the said solution to obtain a concentration of 2% of phenol in the final solution. The crystallization was brought on,

if necessary, by scratching the side of the container with a glass agitator and the crystallization was permitted to go to completeness by standing in darkness at room tembe added to the aqueous solution of dimethyl benzimidazol cobarnide adenosine coenzyme are phenol, hydroxylated phenols, such as resorcinol, hydroquinone, phlorogl-ucinol, etc; hydroxymethylated phenols, such as saligenol; and hydroxy methyl phenols, such as orcinol.

The concentrationof the phenolic compound to be added to the aqueous solution of dimethyl benzirnid'azlol cobazmride adenosine ooenzyme must be below the solubility limit of the phenolic compound in water in order .to form the hydrated crystalline complexes. For example, preferred amounts of some phenolic compounds are perature for a period of about 48 hours. The crystals of the hydrated phenolic complex formed were vacuum filtered, dried over phosphoric acid anhydride, Washed with ether and dried over potassium hydroxide. 88 mg. of a hydrated phenol-dimethyl benzirnidazol cobamide adenosine coenzyme complex were obtained which occurred in the form of red crystals. The crystals were slightly soluble in Water and insoluble for the most part in the usual organic solvents, such as alcohol, ether,

' acetone, benzene and chloroform.

The said complex had the following empirical formula:

72 17 1s ')2 6 5 a 2 )5 and it could be dehydrated under vacuum at 100 C.

of glutamate to }8-' phenol determined by colorimetry and had a purity of 98:1%.

U.V. SPECTRA IN AQUEOUS SOLUTION main, my i fim. after dehydration This product is not described in the literature. Step B.Decomposition of the phenol complex: 80 mg. of the complex of phenol-dimethyl benzimidazol cobamide adenosine coenzyme were dlssolved in 10 parts Dimethyl Benzz-midazb'lcobamide Adnosine C 0 mzym e of acetone containing 50% water. The mixture was heated to 50 C. and the1r80 parts of anhydrous acetone were added thereto. The dimethyl benzimidazol cobamide adenosine coenzyme crystallized and 66 mg. (95% yield) of dimethyl benzimidazol cobamide adenosine coenzyme whose phenol content determined by colorimetry was less than 0.4% and having a purity of 99.5% were obtained."

EXAMPLE II Dimethyl Benzimidazol Cobamide Adenosine Coenzyme Recovery With Phenol From Raw Concentrates 20 liters of an aqueous concentrate of a culture broth of Propionibncterium Shermanii containing about 20 gm.

drated complex of resorcinol-dimethyl benzimidazol cobamide adenosine coenzyme formed were vacuum filtered and dried at room temperature under vacuum in the presence of potassium hydroxide. The dry crystals were washed three times by placing them in suspension in 10 cc. of ether and drying them at room temperature under vacuum in the presence of potassium hydroxide. The hydrated crystalline complex had the following empirical formula and it had a water content of 7.15% (calculated 7.04%

This product is not described in the literature.

Step B.Decompositi0n of the hydrated resorcinol complex: 20 mg. of the hydrated dimethyl benzimidazol cobamide adenosine coenzyme resorcinol complex were dissolved in 10 parts of acetone containing 50% of water. The mixture was heated to 50 C. and then 80 parts of anhydrous acetone were added thereto. Pure dimethyl benzimidazol cobamide adenosine coenzyme was obtained in crystalline form with a yield of 93 EXAMPLE IV Recovery With H yroquinone Step A.Formation of the hydrated hydroquinone complex: 200 mg. of dimethyl benzimidazol cobamide adenosine coenzyme were dissolved in darkness in 20 cc. of aqueous solution containing 5% of hydroquinone, heated to 60 to 70 C. This solution was allowed to stand at room temperature for a period of one week in total darkness. The crystals formed were vacuum filtered and dried at room temperature under vacuum in the presof dimethyl benzimidazol cobamide adenosine coenzyme were obtained by the method of Barker et al. (J. Biol. Chem., vol. 235, 1960, -p 482). The culture broth starting from 152 kg. of moist cellular paste was extracted with ethanol, treated with Dowex 50 Na+, then with Dowex 2 OH: extracted with phenol and fractionated on 1 Dowex 50 at a pH of 3.

10.0 liters of an aqueous solution containing 6% phenol were added to the 20 liters of the said aqueous concentrate and the solutions were mixed to form an aqueous solution having a concentration of 2% phenol. Crystallization of the hydrated complex of phenol-dimethyl benzimidazol cobamide adenosine coenzyme was induced by scratching the side walls of the container and the solution was allowed to stand in the dark at room temperature for 72 hours to complete the crystallization. The crystals formed were vacuum filtered and washed with an aqueous solution containing 2% phenol.

The washed crystals were then added to a aqueous acetone solution heated to 50 C. and maintained at this temperature for several minutes until complete solution of the crystals occurred. After the addition of 80 parts of acetone to the solution, 17.5 gm. of dimethyl benzimidazol cobamide adenosine coenzyme crystallized and the said crystals were determined by spectrophotometry to be 96 to 100% pure.

EXAMPLE III Dimethyl Benzimidazol cobamide Adenosine Coenzyme Recovery With Resorcinol Step A.Formation of the hydrated resorcinol complex: 100 mg. of dimethyl benzimidazol cobamide adenosine coenzyme were dissolved in darkness in 5 cc. of distilled water heated to 50 C. 5 cc. of an aqueous soluence of potassium hydroxide. The dried crystals were washed three times by placing them in suspension in 10 cc. of ether and drying them at room temperature under vacuum in the presence ofpotassium hydroxide. 205 mg. of the hydrated dimethyl benzimidazol cobamide adenosine coenzyme hydroquinone complex were obtained in the form of red crystals.

The complex was soluble in water in an amount of about 2 parts per thousand. It was decomposed by dilute aqueous alkalis and was slightly soluble in ethanol and insoluble in ether, acetone, benzene and chloroform. The complex product had the following empirical formula:

EXAMPLE V Dimethyl Benzimidazol Cobamide Adenosine Coenzyme Recovery With Saligenol Step A.Formation of the hydrated saligenol complex: mg. of dimethyl benzimidazol cobamide adenosine coenzyme were dissolved in total darkness in 2 cc. of distilled water heated to 50 C. 8 cc. of an aqueous solution containing 5% of saligenol were added to the solution obtained. The solution was allowed to stand for a period of one week in total darkness at room temperature to effect crystallization. The crystals formed were vacuum 'filtered and dried under vacuum at room temperature in the presence of potassium hydroxide. The crystals were washed three times by placing them in suspension in 10 cc. .of ether and drying them at room temadenosine coenzyme saligenol complex was obtained in the form of red crystals.

The complex was slightly soluble in water (about 2 parts per thousand). It was decomposed by dilute aqueous alkalis and was slightly soluble in dilute aqueous acids and in ethanol and insoluble in acetone, benzene and chloroform. The hydrated complex had the following empirical formula:

The water content was 4.9% (calculated 4.85%). This product is not described in the literature.

. Step B.-Dec0mp0sition of the hydrated saligenol complex: 40 mg. of the hydrated dimethyl benzimidazol cobamide adenosine coenzyme saligenol complex were dissolved in parts of 50% aqueous acetone heated to 50 C. and then 80 parts of anhydrous acetone were added thereto. Pure dimethyl benzimidazol cobamide adenosine coenzyme crystals were obtained with a yield of 95%.

Various modifications of the process of the invention may be made without departing from the spirit or scope thereof and it is to be understood that the invention is 'intended to be limited only as defined in the appended claims.

We claim: a 1. Hydrated crystalline dimethyl benzimidazol cobamide adenosine coenzyrne-phenolic complexes having the formula wherein R is a phenol having the formula in which R is at least one member selected from the group consisting of hydroxy, lower alkyl and hydroxy lower alkyl, x is an integer from O to 2, m is an integer from 2 to 10 and n is an integer from v1 to 3.

in which R is at' least one member selected from the group consisting of hydroxy, methyl and hydroxymethyl, x is an integer from O to 2, m is an integer from 2 to 10 and n is an integer from 1 to 3.

3. A hydrated crystalline dimethyl benzimidazol cobamide adenosine coenzyme-phenol complex having the formula '12 1oo 17 1s )2 e s a 2 5 4. A hydrated crystalline dimethyl benzimidazol cobamide adenosine coenzyme-resorcinol complex having the formula C'YZHIOOOI'INIBPCO' s 4( )2]s' 2 18 5. A hydrated crystalline dimethyl benzimidazol cobamide adenosine coenzyme-hydroquinone complex having the formula 6. A hydrated crystalline dimethyl benzimidazol cobamide adenosine coenzyme-saligenol complex having the formula References Cited in the file of this patent UNITED STATES PATENTS Bernhaver et a1. Feb. 11, 19 58 Nomine et al. May l6, 1961 

1. HYDRATED CRYSTALLINE DIMETHYL BENZIMIDAZOL COBAMIDE ADENOSINE COENZYME-PHONOLIC COMPLEXES HAVING THE FORMULA C72H100O17N18PCO$(R)N$(H2O)M 